After isolating the MNCs using Ficoll, you may further enrich monocytes through a Percoll density gradient.

Please see

http://www.ncbi.nlm.nih.gov/pubmed/12957415

http://www.ncbi.nlm.nih.gov/pubmed/6259262

Hi, I had better results by using serum free DMEM medium. Adherence time shows high variability between donors, normally 30-120 min is required.  Longer incubation times cause partial activation on monocytes (especially when there is LPS contamination in the reagents, and this may results in Monocyte-Tcell clamps).

Macrophages are quite hard to detach after differentiation. Therefore, in our lab, we routinely eliminate non-monocytic cells from macrophage cultures with a fast incubation (2 minutes) with trypsin-EDTA 1X (0.05%) at room temperature, twenty four hours after cells are plated. 

In my opinion your seeding cell concentration is too low. I use >10E7 cells/ml when I plate monocytes on plastic (typically >100x10E6 per T75 flask). Monocytes preferentially adhere to plastic, but if you leave too much 'empty space', lymphocytes will attach as well, so you need to have enough monocytes to reach confluence so that those will displace any lymphocyte that might want to attach.

The adherence of the monocytes will also be strongly influenced by your medium. Using serum free media (such as AIM-V or X-VIVO15) will give you a slightly different yield than using RPMI with fetal calf serum, and this will again be different when using human serum/plasma. In any case, 2 hours is plenty for the primary adherence step.

Thank you all for your helpful suggestions!

Olivier, I will certainly try seeding at a greater density. 10E6 or 10E7 per ml indeed is a big difference.

Korcan, Fernando, Olivier: I use RPMI160 without serum since the next manipulation occurs using this medium and I would prefer to avoid switching media. However, certainly worth a try if things keep slacking.

Bernd, why use warm PBS-EDTA? We always use cold in our lab, so maybe we need to change this then.

Lucio, I did not know macrophages adhere so hard they resist a fast incubation with trpsin-EDTA. Thanks for the tip. Does this also count for monocytes then?

Korcan, doe the partial activation of monocytes caused by longer incubation times only apply to the adherence enrichment step or also to subsequent incubation after removal of other cell types?

Pramod, I would rather use adherence for monocyte enrichment. Thanks anyway for the potentially useful alternative method.

Fernando, I am certainly interested in your old/inexpensive method for T cell depletion from PBMC. I was planning to do this for another experiment using magnetic isolation, but off course we prefer less expensive methods is equally satisfying results can be obtained.

Thanks again, this will definitely help me in the lab.

Jorrit,

In our projects we have to make sure that HIV DNA/RNA observed in cultured macrophages are not coming from contaminant T cells, which can be confirmed using a sensitive PCR for the detection of TCR mRNA. We tried several protocols for the elimination of T cells (positive and negative selection; Percol separation), but the best results came from the fast trypsinization I suggested. Macrophages are very hardy and will stay adherent even after a 5 min trypsin treatment at RT. We found out that 1 to 2 minutes is enough to remove non-adherent cells that otherwise would be attached to the macrophages themselves.